Methods for selective inhibition of pluripotent stem cells

ABSTRACT

Provided herein are methods of reducing or eliminating undifferentiated pluripotent stem cells, where the methods comprise contacting an effective amount of a compound to a heterogeneous cell population or sample comprising or suspected of comprising differentiated cell types and undifferentiated pluripotent stem cells, whereby the contacting selectively reduces or eliminates undifferentiated pluripotent stem cells from the cell population or sample. Also provided are methods for obtaining a population of stem cell-derived cell types substantially free of undifferentiated pluripotent stem cells as well as isolated populations of such of stem cell-derived cell types.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/929,659, filed Jan. 21, 2014, which is incorporated by reference asif set forth in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.4R00HL094708-03, awarded by the National Heart, Lung, and BloodInstitute and the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND

Pluripotent stem cells, which include induced pluripotent stem cells(iPSCs) and embryonic stem cells (ESCs), are characterized by a capacityfor self-renewal and an ability to differentiate into any functionalcell in the body. Thus, pluripotent stem cells are valuable sources ofdifferentiated somatic cell types for research and clinicalapplications. The advent of human iPSCs (hiPSCs), derived from somaticcells by the exogenous expression of defined transcription factors, hasovercome ethical issues associated with human ESCs (hESCs) and, whenderived from the patient, may avoid immunological complications. Whilepromising, significant limitations to the therapeutic use of hiPSCsremain unresolved. These include interline variations ranging frominconsistent transcription factor expression and differential DNAmethylation to sporadic point mutations and chromosomal defects thataffect in vitro differentiation, tumorigenicity, and potential clinicalapplications (Lee et al., Nature Med. 19(8): p. 998-1004 (2013);Robinton and Daley, Nature 481:295-305 (2012); Feng et al., Stem Cells28:704-712 (2010); Gore et al., Nature 471:63-67 (2011)). Moreover,current tests of hiPSC potency rely on extensive in vitrodifferentiation tests, in vivo teratoma assays in rodents (Robinton andDaley, Nature 481,295-305 (2012); Maherali and Hochedlinger, Cell StemCell 3:595-605 (2008)) or bioinformatic and gene expression assays (Bocket al., Cell 144:439-452 (2011); Muller et al., Nat. Methods 8:315-317(2011)) which cannot be practically implemented into high-throughputhiPSC line generation designed to limit interline variability.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of reducing oreliminating undifferentiated pluripotent stem cells. In one embodiment,the method comprises contacting an effective amount of a compound to aheterogeneous cell population or sample comprising or suspected ofcomprising differentiated cell types and undifferentiated pluripotentstem cells, whereby the contacting selectively reduces or eliminatesundifferentiated pluripotent stem cells from the cell population orsample. The compound can be STF-31, FK866, or other inhibitor ofnicotinamide phosphoribosyltransferase (NAMPT). The effective amount canbe an amount between about 0.1 μM and about 100 μM.

In a further aspect, the present invention provides a method ofobtaining a population of stem cell-derived cell types substantiallyfree of undifferentiated pluripotent stem cells. In one embodiment, themethod comprises (a) inducing undifferentiated pluripotent stem cells todifferentiate or partially differentiate into one or more stemcell-derived cell types; (b) contacting an effective amount of acompound to the induced cell population, whereby the contactingselectively reduces or eliminates undifferentiated pluripotent stemcells from the induced cell population; and (c) isolating the contactedcells to obtain a population of one or more stem cell-derived celltypes, wherein the population is substantially free of undifferentiatedpluripotent stem cells. The compound can be STF-31. The effective amountcan be an amount between about 0.1 μM and about 100 μM. In some cases,the effective amount is about 2.5 μM. The one or more stem cell-derivedcell types can be a cardiomyocyte, neural progenitor, neuron, retinalpigmented epithelial cell, liver cell, or mesenchymal stem cell.

In some embodiments, the method can further comprise expanding theisolated stem cell-derived cell types as single cell clones.

In another aspect, the present invention provides a population of stemcell-derived cell types substantially free of undifferentiatedpluripotent stem cells obtained by a method provided herein.

These and other features, aspects, and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention. Thedescription of preferred embodiments is not intended to limit theinvention to cover all modifications, equivalents and alternatives.Reference should therefore be made to the claims recited herein forinterpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents data collected for human pluripotent stem cells (hPSCs)treated with small molecules. (A) Representative bright field images ofconfluent human induced pluripotent stem cells (hiPSCs) (DF6-9-9T) aftertreatment with 2.5 μM STF-31, 30 μM WZB117, and 20 μM PluriSIn for 24-72hours. Scale bar=50 μm. (B) Cell viability as measured by SYTOX greenassay in hiPSC treated with 20 μM PluriSIn, 30 μM WZB117, and 2.5 μMSTF-31 (N=3). (C) Representative images of live (calcein AM-green)/deadstaining (ethidium homodimer 1-red) in hiPSC after treatment with 20 μMPluriSIn, 30 μM WZB117, and 2.5 μM STF-31. Scale bar=100 μm. (D)Titration of STF-31 for 24-72 hours in confluent hiPSC determined byneutral red assay (N=3). (E) Cell viability of varying densities ofhiPSC after treatment with 20 μM PluriSIn, 30 μM WZB117, and 2.5 μMSTF-31 determined by neutral red assay (N=3). Representative images ofcell morphology and density at 24 hours and 96 hour post-plating arecoupled with respective bar graphs. Scale bar=200 μm. (F) Percent oflive hiPSC in each phase of the cell cycle at 24-96 h post-plating(N=3). Schematic and results of colony forming assay where cells werepassaged (G) or washed (H) prior to continued culture for 6 days priorto alkaline phosphatase staining. Arrows indicate individual colonies.Bar graphs representing average number of colonies per plate for eachtreatment scheme are shown (N=3). Data are represented as mean±SEM. *p≤0.05, ** p≤0.01, *** p≤0.001 compared to DMSO control.

FIG. 2 presents data obtained for twenty-four hour pulse treatment forselective in vitro elimination of hiPSC. Titration (A) and comparison ofsmall molecules (B) following 24 hours of treatment with STF-31(0.01-100 μM), 30 μM WZB117, and 20 μM PluriSIn in sub-confluent andconfluent hiPSC (DF6-9-9T), fibroblasts, and DF6-9-9T hiPSC-derivedcardiomyocytes. Average viability was measured with neutral red assay 72hours after treatment initiation (N=3). Representative flow cytometryhistogram (C) of TNNI3 and IRX4 abundance in cardiomyoyctes 72 hoursafter initiation of a 24 hour treatment with 5 μM STF-31 (N=3).Representative images (D) for immunofluorescent detection of TNNT2organization in day 15 cardiomyocytes following a 24-hour pulsetreatment with 5 μM STF-31 on day 10 of differentiation. Top paneldisplays cell clusters imaged at 40× and the lower panel has individualcells showing structural organization imaged at 100× magnification. Thescale bar in the top panel is 50 μm and the bottom panel is 20 μm (N=3).Bar graphs (E) for qPCR of TNNI3, TNNT2, and NKX2.5 24-72 hours afterinitiation of a pulse treatment with 5 μM STF-31 in day 10cardiomyocytes. Representative images of alkaline phosphatase stainingto detect DF6-9-9T hiPSC colonies in co-cultures with day 10cardiomyocytes (F) and human fibroblasts (G). DF6-9-9T were platedbetween 1e2 and 1e4 live cells and treated for 48 hours with 5 μM STF-3124 hours after plating (N=3 for cardiomyocytes and N=5 for humanfibroblasts). Data are represented as mean±SEM. ** p≤0.01compared toDMSO control.

FIG. 3 presents data demonstrating STF-31 inhibits NAD⁺ salvage pathway.(A) Effect of glucose deprivation on hiPSC (DF6-9-9T) viability asmeasured by neutral red assay in sub-confluent cells and confluent cells(N=3). (B) ECAR and OCR in hiPSC after treatment with 2.5 μM STF-31 and30 μM WZB117 (N=3). (C) Bar graphs representing glucose uptake as apercentage of vehicle control for hiPSC treated for 1, 15, 18, and 24hours with 2.5 μM STF-31, 30 μM WZB117, and 20 μM cytochalasin B in E8media (N=3). (D) Representative immunoblots for total and cleavedcaspase-9, cleaved caspase-3, and GAPDH in sub-confluent hiPSC treatedwith glucose deprivation, 30 μM WZB117, or 2.5 μM STF-31. hiPSC weretreated for 3 hours with 1 μM camptothecin as a positive control forimmunoblotting. (E) Nucleotide levels in hiPSC after treatment with 20mM 2-DG for 24 h or 2.5 μM STF-31 for 3-24 h (N=3). (F) hiPSC viabilityas measured by neutral red assay in cells treated with STF-31 alone orin the presence of 1 or 10 μM nicotinic acid (N=3). Above each bar arerepresentative brightfield images illustrating cell density andmorphology for 48-hour treatment in each condition. Data are representedas mean±SEM. * p≤0.05, *** p≤0.001 compared to media or DMSO control.

FIG. 4 is a diagram representing temporal effects of treating hPSCs (A)with STF-31 or (B) glycolytic inhibition.

FIG. 5 presents data demonstrating that STF-31 is toxic to humanembryonic stem cells (hESCs) when cultured in the presence offibroblasts. (A) Representative images of alkaline phosphatase staining(top panel) and brightfield imaging (middle panel) of H1 hESC coloniesgrown on mitotically inactivated human fibroblast feeders after 96 hoursof continuous treatment with 2.5 μM STF-31. Representative images ofstaining for alkaline phosphatase activity in H1 colonies. 72 hoursafter passaging colonies treated with vehicle control or STF-31 for 96hours with 2.5 μM STF-31 (N=3). (B) Representative density plots ofco-staining for BrDU incorporation and 7-AAD in discontinuouslyproliferating hiPSC 24-96 hours post-plating. * p≤0.05, *** p≤0.001compared to DMSO control. (C) Representative images of alkalinephosphatase staining to detect DF6-9-9T hiPSC colonies in co-cultureswith day 10 cardiomyocytes. DF6-9-9T were plated between 1e2 and 1e4live cells and treated for 24 hours with 5 μM STF-31 24 hours afterplating (N=2).

FIG. 6 demonstrates the effects of media, timing, and concentration onPluriSIn- and STF-31-mediated toxicity. (A-B) Bar graphs representingcell viability in E8 media after 24-96 h treatment with 20-40 μMPluriSIn and 2.5 μM STF-31 as measured by neutral red assay in confluenthiPSC (DF6-9-9T; a) and hESC (H1; b) (N=3). (C-D) Bar graphsrepresenting cell viability in mTeSR media after 24-96 hours treatmentwith 20 μM PluriSIn and 2.5 μM STF-31 as measured by neutral red assayin confluent hiPSCs (c) and hESCs (d) in mTeSR media (N=3). * p≤0.05, **p≤0.01, *** p≤0.001 compared to DMSO control.

FIG. 7 demonstrates the effects of glucose deprivation, STF-31, andWZB117 on metabolic flux and expression of apoptosis markers. (A) ECARand OCR in hiPSC (DF6-9-9T) treated with 20 mM 2-deoxy-d-glucose (2-DG).(B) Representative immunoblots for p-AMPK and AMPK levels insub-confluent hiPSC treated for 6-36 hours with glucose deprivation, 30μM WZB117, and 2.5 μM STF-31 (N=3). (C) Bar graph representingcaspase-3/7 activity in sub-confluent hiPSC treated with 2.5 μM STF-31,30 μM WZB117, and glucose deprivation for 6-24 hours as measured by afluorescence based caspase-3/7 activity. * p≤0.05, *** p≤0.001 comparedto media or DMSO control. (FIGS. 7D-E) Densitometry for immunoblottingof p-AMPK, cleaved caspase-3, and cleaved caspase-9 in sub-confluentcells corresponding to blots in FIG. 3D. (D) Densitometry for 6-36 hourglucose deprivation in hiPSC (N=3). (E) Densitometry for 2.5 μM STF-31and 30 μM WZB117 6-36 hour treatment (N=3).

FIG. 8 demonstrates that STF-31 is toxic to pluripotent stem cells, buthiPSC-derived progeny and terminally differentiated cells are resistantto STF-31 toxicity. (A) Phase contrast images of hESCs, hiPSCs, anddifferentiated cells upon STF-31 treatment (2.5 μM for 72 hours). (B)Human iPSC-derived cardiomyocytes were treated on day 10 ofdifferentiation with 2.5 μM STF-31, and protein and mRNA levels forcardiomyocyte markers were assessed 72 hours later by flow cytometry(top), immunofluorescence (middle), and 24-72 hours later by qRT-PCR(bottom). (C) Human iPSC-derived neural progenitors were treated with2.5 μM for 72 hours and assessed by MTT assay (top; day 10 and day 12progenitors) and immunofluorescence (bottom; day 12 progenitors shown).In all cases, STF-31-treated cardiomyocytes and progenitor cells wereindistinguishable from controls.

FIG. 9 presents the chemical structures of (A) STF-31 and (B) FK866, anon-competitive inhibitor of NAMPT.

FIG. 10 presents data regarding FK866 toxicity on human pluripotent stemcells. (A) Bar graph demonstrates that FK866 is toxic to humanpluripotent stem cells. Cell viability data is presented for hiPSCstreated with FK866 for up to 72 hours. Timing of cell death for thisNAMPT inhibitor was similar to that observed for STF-31, independent ofconcentration. (B) Cell viability of human pluripotent stem cells 72hours after treatment initiation with FK866. Cells were treated with arange of concentrations (1 nM to 10 μM) either continuously or for 24hour pulse.

FIG. 11 presents data demonstrating adult pigmented retinal epithelialcells (ARPE) and PAX6 positive neural progenitor cells are resistant toSTF-31 treatment. (A) Brightfield images of human embryonic stem cells(hESC), human induced pluripotent stem cells (hiPSC), and ARPE19 treatedwith 2.5 μM STF-31 for up to 72 hours, showing no adverse effects onARPE cell morphology or viability. 5 μM STF-31 shows similar results(not shown). (B) Cell viability of H7 hESC, neural progenitor cells (day10,12, 14 of differentiation), and ARPE19 treated with 2.5 μM STF-31 for24 hours. Data expressed as mean+S.D. (n=4); p<0.05, ** p<0.01, ***p<0.001. (C) Cell viability of PAX6 positive neural progenitor cellstreated with STF-31 and nicotinic acid (NA) for 72 hours. Datademonstrate that the cell death observed in neural progenitor cellstreated for 72 hours continuously with STF-31 can be rescued withaddition of exogenous NA, consistent with the mechanism of NAMPTinhibition observed in the pluripotent stem cells. Data expressed asmean+S.D. (n=4), * p<0.05, ** p<0.01, *** p<0.001. Altogether, thesedata demonstrate that short term treatment (24 hours) of the neuralprogenitor and ARPE cells does not result in significant toxicity.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth in the present application.

The invention described herein is based, at least in part, on theInventors' discovery that pluripotent stem cells can be selectivelyeliminated from a mixed population of differentiated andundifferentiated pluripotent cells. Terminally differentiated adultcells and partially differentiated ESCs and iPSCs generally rely onoxidative phosphorylation for cellular metabolism and growth, whereasundifferentiated ESCs and iPSCs are glycolysis-dependent and expressseveral genes that encode various glucose transporter proteins formediating glucose uptake. Using microarrays and proteomics, theInventors identified a discrepancy in the correlation betweentranscription and cell surface localization of several glucosetransporter proteins (e.g., GLUT1, GLUT3, and GLUT4). While WZB117eliminates hPSC through inhibition of GLUT1, the Inventors discoveredthat STF-31, which was previously considered to be a GLUT1 inhibitor,does not function in this manner and, instead, removes hPSC throughinhibition of the nicotinamide adenine dinucleotide (NAD⁺) salvagepathway. These discoveries are consistent with and supported by twosubsequent reports. Adams et al., ACS Chem. Biol. 9(10):2247-2254(2014); Dragovich et al., Bioorg Med Chem Lett 24:954-962 (2014). Basedon these findings, we developed a novel strategy for using STF-31 toeffectively eliminate human pluripotent stem cells across a range ofculture conditions by a process that spares differentiated progeny.These results provide an important advancement towards the developmentof clinically safe hPSC-derived progeny for human stem cell basedtherapies.

Accordingly, provided herein are methods for eliminating pluripotentstem cells from mixed cell populations of differentiated cells. In afirst aspect, the present invention provides methods for obtaining acell population devoid of undifferentiated pluripotent cells. Inparticular, the invention described herein provides methods forobtaining populations of cells differentiated from pluripotent stemcells, where the populations are free or substantially free ofundifferentiated and potentially tumorigenic pluripotent stem cells.Methods of the present invention include methods of reducing oreliminating such undifferentiated pluripotent stem cells from a cellpopulation comprising differentiated and undifferentiated cell types. Asused herein, the terms “free” and “substantially free” with regard tocell populations refer to cell populations that comprise fewer thanabout 5%, preferably fewer than about 1%, and more preferably fewer thanabout 0.1% (e.g., fewer than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%,0.01%, 0%) undifferentiated pluripotent stem cells per unit volume, ascompared to the cell population from which it was obtained. Cellpopulations obtained according to a method provided herein can beassessed and identified as free or substantially free ofundifferentiated pluripotent stem cells using, for example, visualexaminations (e.g., visually identifying residual undifferentiatedpluripotent cells in a contacted cell population, quantitative real-timepolymerase chain reaction (qRT-PCR) (e.g., monitor the absence orreduction in the gene expression of pluripotency markers), or flowcytometry (e.g., monitor the absence or reduction in the protein levelof pluripotency markers). As used herein, the term “substantiallydifferentiated” cell population refers to a population of cellscontaining at least about 50%, preferably at least about 60%, 70%, or80%, and even more preferably, at least about 90%, differentiated cellsrepresenting one or more stem cell-derived cell types.

In general, methods of the invention are effected by contacting a cell,cell population, or sample with a chemical or compound according to theinvention. More particularly, methods of the present invention caninclude contacting an effective amount of a chemical or compound to aheterogeneous cell population that comprises undifferentiatedpluripotent stem cells and differentiated cell types derived frompluripotent stem cells, whereby the contacting selectively reduces oreliminates undifferentiated pluripotent stem cells from the cellpopulation or sample. Chemicals and compounds appropriate for useaccording to a method provided herein exhibit selectivity in inhibitingor eliminating pluripotent stem cells without damaging other (e.g.,differentiated) cell types. A chemical or compound that “selectively”inhibits or eliminates an undifferentiated pluripotent stem cell is achemical or compound that inhibits the viability or promotes the deathof an undifferentiated pluripotent stem cell, but does not substantiallyinhibit the viability or promote the death of a differentiated celltype. As used herein, the term “inhibits,” “inhibition,” or “inhibiting”refer to a decrease by any value between 10% and 90%, or of any integervalue between 30% and 60%, or over 100%, or a decrease by 1-fold,2-fold, 5-fold, 10-fold, or more. As used herein, the terms “promote” or“promoting” refer to an increase by any value between 10% and 90%, or ofany integer value between 30% and 60%, or over 100%, or an increase by1-fold, 2-fold, 5-fold, 10-fold, or more.

Any appropriate chemical or compound can be used according to thepresently described methods. In some embodiments, a chemical or compoundappropriate for use according to a method provided herein is capable ofsuppressing the activity or expression of NAD⁺ (nicotinamide adeninedinucleotide), a coenzyme that plays a critical role in manyphysiologically essential processes (Ziegkel, Eur. J. Biochem.267:1550-1564 (2000)). NAD⁺ synthesis is mediated by NAMPT (nicotinamidephosphoribosyltransferase), an enzyme that converts nicotinamide tonicotinamide mononucleotide, which is converted into nicotinamideadenine dinucleotide (NAD+) by nicotinamide mononucleotideadenylyltransferase in the mammalian biosynthetic pathway. Since NAMPTis the rate-limiting factor in NAD⁺ biosynthesis, NAMPT inhibitorsdeplete intracellular NAD⁺ levels. Small-molecule NAMPT inhibitorsinclude, without limitation, STF-31 (Chan et al., Sci Transl Med.3:94ra70 (2011)); FK866 (also known as APO866); GNE-617 (N-{[4-(35-difluorobenzenesulfonyl)phenyl]methyl}imidazo[1,2-a]pyridine-6-carboxamide);GNE-618(N-(4-((3-(trifluoromethyl)phenyl)-sulfonyl)benzyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxamide); and GMX-1778 (also known as CHS828).

FK866 has been shown to selectively block proliferation and induceapoptosis of activated T cells (Busso et al., Plos One 3:e2267 (2008)).STF-31 (also known as4-[[[[4-(1,1-Dimethylethyl)phenyl]sulfonyl]amino]methyl]-N-3-pyridinyl-benzamide)has the empirical formula C₂₃H₂₅N₃O₃S, is soluble in DMSO, and iscommercially available from distributors such as Tocris, EMD Millipore(Merck KGaA). GNE-617 was previously described by Zheng et al., J MedChem. 56:6413-6433 (2013). GNE-618, which is structurally related toGNE-617, was previously described by Zheng et al., Bioorg Med Chem Lett.23:5488-5497 (2013). GMX-1778 has been described as a potent andspecific inhibitor of the NAD⁺ biosynthesis enzyme NAMPT (Watson et al.,Molecular and Cellular Biol. 29(21): 5872-5888 (2009)). See also vonHeideman et al., Cancer Chemother. Pharmacol. 65(6):1165-72 (2010).Other NAMPT inhibitors appropriate for the methods provided hereininclude, without limitation, those compounds having structuralsimilarity to STF-31 (see International Application No.PCT/US2012/022113) or GNE-617/618 as described in Dragovich et al.,Bioorg Med Chem Lett. 24(3):954-62 (2014); Zheng et al., J Med Chem,56(16):6413-33 (2013); and Zheng et al., Bioorg Med Chem Lett,23(20):5488-97 (2013). For example, compounds having structuralsimilarity to STF-31 for use according to a method provided hereininclude, without limitation,4-(Phenylsulfonamidomethyl)-N-(quinolin-5-yl)benzamide (Vitas,STK647082); 4-tert-Butyl-N-(4-(pyridin-3-ylcarbamoyl)benzyl)benzamide(Otava Chemicals, 6360023); and4-((4-Methoxyphenylsulfonamido)methyl)-N-(pyridin-3-yl)benzamide (Vitas,STK643640).

An effective amount of a chemical or compound for use according to amethod provided herein can be a concentration betwee about 1 nanomolar(nM) to about 100 micromolar (μM) (e.g., about 0.5 nM, 1 nM, 5 nM, 10nM, 15 nM, 20 nM, 25 nM, 30 nM, 50 nM, 75 nM, 90 nM, 0.1 μM, 0.5 μM, 1μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 10 μM, 15μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM). Insome cases, an effective amount of a chemical or compound for useaccording to a method of the invention is a concentration of about 1 μMto about 2.5 μM (e.g., about 1, about 1.5, about 2, about 2.5 μM). Asused herein, an effective amount is an amount of a chemical or compoundcapable of eliminating pluripotent stem cells (e.g., hPSCs). Thehalf-maximal effective concentration can be presented as an EC₅₀ value.An effective amount of STF-31 is about 2.5 μM, which is substantiallyless than effective amounts of either benzethonium chloride ormethylbenzethonium chloride (e.g., greater than 5 μM compared to 1 μMSTF-31). An effective amount of FK866 is about 1.0 nM to about 25 nM(e.g., about 0.5 nM, 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM). Selectiveinhibition of pluripotent stem cells has been described followingexposure to 20 μM PluriSin #1 (Ben-David et al., Cell Stem Cell12(2):167-179 (2013)).

Chemicals and compounds appropriate for use according to a methodprovided herein can be contacted to a cell, cell population, or samplefor any appropriate length of time. For example, an effective amount ofa chemical or compound capable of eliminating pluripotent stem cells canbe contacted to a heterogeneous cell population for a time sufficient toensure elimination of all pluripotent stem cells from the cells, cellpopulation, or sample. In some cases, a sufficient length or period oftime is at least 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours,60 hours, 72 hours, or 96 hours. For example, cells can be exposed to achemical or compound such as STF-31 or FK866 for about 72 hours plussupplementation with compound in fresh culture medium every 24 hours.There can be an inverse relationship between the effective amount ofchemical or compound used and the length of time required to selectivelyeliminate pluripotent stem cells. In other words, the length of exposurecan decrease as the effective amount of, for example, the compound(e.g., STF-31, FK866) increases.

The effectiveness of a chemical or compound in eliminatingundifferentiated pluripotent stem cells from a cell population can beconfirmed (qualitatively or quantitatively) by detecting the presence orabsence of undifferentiated pluripotent stem cells, assessing expressionof differentiated cell type-specific markers, or determining cellviability following contacting or exposure to a chemical or compoundthat selectively inhibits or eliminates undifferentiated pluripotentstem cells. RNA or proteins can be extracted from the cells and assayed(via Northern hybridization, RT-PCR, Western blot analysis, etc.) forthe presence of markers indicative of a desired phenotype. For example,pluripotent stem cells can detected by various cell surface markers,including SSEA-3, Tra-1-60, and Tra-1-81. SSEA-3⁺ cells can be detectedusing a chromophore-conjugated antibody having specificity to thatparticular antigen. In some cases, one or more cell surface markerscorrelated with an undifferentiated state (e.g., SSEA-4, Tra-1-60, andTra-1-81), as well as the pluripotent stem cell transcription factormarker, Oct-4, can be detected. Also, undifferentiated pluripotent stemcells have typical stem cell morphology, which is well described in theart.

Differentiated cell types such as cardiomyocytes can be identified usingqRT-PCR to detect expression of cardiac markers such as NKX2.5 or TNNT2.Adult multipotent stem cells can be identified based upon highexpression levels of the enzyme aldehyde dehydrogenase (ALDH). Ofcourse, cells can be assayed immunohistochemically or stained, usingtissue-specific stains. In other cases, telomere length can be used asan indicator of differentiation. In general, undifferentiated stem cellshave longer telomeres than differentiated cells; thus the cells can beassayed for the level of telomerase activity.

Cell viability can be assessed by measuring a percentage of viable cellsfollowing one or more exposures to a compound such as STF-31. Cellviability can be demonstrated by various methods including, withoutlimitation, exclusion of a vital dye such as trypan blue or7-amino-actinomycin D (7-AAD), loss of uptake of neutral red(3-Amino-7-dimethylamino-2-methylphenazine hydrochloride), or reductionof the tetrazolium dye MTT dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide to insoluble formazan.

In some cases, a cell population obtainable by a method provided hereinis a stem cell-derived cell population devoid of, or substantiallydevoid of, undifferentiated pluripotent stem cells. Pluripotent stemcells include induced pluripotent stem (iPS) cells and embryonic stem(ES) cells, both of which are valuable sources of differentiated somaticcell types for research and clinical applications. Cells of a populationobtainable by a method provided herein, therefore, can be multipotent,oligopotent, unipotent, or terminally differentiated cell types. As usedherein, “multipotent cells” include cells and their progeny, which maybe able to differentiate into, or give rise to, multipotent, oligopotentand unipotent progenitor cells, and/or one or more mature or partiallymature cell types, except that the mature or partially mature cell typesderived from multipotent cells are limited to cells of a particulartissue, organ or organ system. Multipotent stem cells include, withoutlimitation, hematopoietic stem cells, neuronal stem cells, and bonemarrow stem cells. Oligopotent stem cells include, without limitation,intestinal stem cells, mammary stem cells, myeloid or lymphoid precursorcells, cells from amniotic fluid, mesenchymal progenitorcells/multipotent stromal cells/mesenchymal stem cells (MSCs),glial-restricted precursor cells, bipotential precursor cells from fetalliver, peripheral blood mononuclear cells, mast cell precursor cells,satellite cells, dermal stem cells, hair follicular stem cells, basalstem cells, hematopoietic cells, myeloid precursor cells, mesenchymalprogenitor cells, glial-restricted precursor cells, bipotentialprecursor cells from fetal liver, umbilical cord blood cells, peripheralblood stem cells, cells from amniotic fluid, bone marrow stem cells, andmast cell precursor cells.

In some cases, a cell population obtainable by a method provided hereinis substantially homogeneous, consisting essentially of a single stemcell-derived cell type (e.g., cardiomyocytes, neurons, liver cells,neural stem cells, hematopoetic stem cells). Any appropriate method canbe performed to detect the presence of undifferentiated pluripotent stemcells in a cell population obtained according to a method providedherein. For example, assays can be performed to assess the potential fora cell population or subset thereof to form tumors comprising cellsderived from all three germ layers such as teratomas, teratocarcinomas,or other germ cell tumors. In some cases, a teratoma assay includesinjecting 1e3-5e6 human cells into a tissue or organ of an animal.kidney, testis, intramuscular, or subcutaneous. According to someteratoma assay protocols, tumors resulting from the injection areassessed by certified pathologist 6-12 weeks after transplantation oronce tumor diameter reaches defined endpoint. For review, seeWesselschmidt, R. L., Methods Mol Biol. 767:231-41 (2011); Zhang et al.,Teratoma formation: A tool for monitoring pluripotency in stem cellresearch, in Stem Book 2008: Cambridge (Ma.).

Differentiated cell types appropriate for cell populations of thepresent invention include any differentiated cell type of the three germlayers, endoderm, mesoderm, and ectoderm. In some cases, differentiatedcells include, without limitation, stem cell-derived cardiomyocytes,neural progenitors, neurons, vascular smooth muscle cells (obtained frommultiple lineages), endothelial cells, neurons, retinal pigmentedepithelial cells, liver cells, and mesenchymal stem cells.

In another aspect, the present invention provides an isolated populationof stem cell-derived cells obtained by a method described herein.Populations of stem cell-derived cell types that are free orsubstantially free of undifferentiated pluripotent stem cells are usefulfor research and clinical applications, including tissue engineering.

In a further aspect, the present invention provides therapeuticformulations comprising one or more compounds that selectively reduce oreliminate undifferentiated pluripotent stem cells from a cellpopulation. Preferably, a therapeutic formulation provided herein willbe formulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal (e.g., human)being treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The compound (e.g., NAMPT inhibitor)need not be, but is optionally formulated with one or more agentscurrently used to prevent or treat cancer or to treat or prevent anotherclinical disease or disorder. Preferably, a therapeutic formulationprovided herein is prepared by mixing one or more compounds thatselectively reduce or eliminate undifferentiated pluripotent stem cellsfrom a cell population with a physiologically acceptable carrier,excipient, or stabilizer. In exemplary embodiments, the therapeuticformulation is prepared in the form of a lyophilized formulation oraqueous solution.

Also provided herein are methods comprising administration oftherapeutically effective amount of a compound or therapeuticformulation thereof to a subject in need thereof. As used herein, a“therapeutically effective amount” of the compound or therapeuticformulation to be administered will be an amount necessary to depletesubstantially all pluripotent cells from a complex mixture; or todecrease the number of teratoma cells in a subject. In exemplaryembodiments, chemicals, compounds, and formulations of the presentinvention are useful in vitro and in vivo for depletion of pluripotentstem cells, including the prevention of teratoma formation uponadministration of a stem cell-derived cell population provided herein.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN.™, PLURONICS.™. or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

The therapeutic dose may be at least about 0.01 μg/kg body weight, atleast about 0.05 g/kg body weight; at least about 0.1 g/kg body weight,at least about 0.5 g/kg body weight, at least about 1 μg/kg body weight,at least about 2.5 g/kg body weight, at least about 5 μg kg body weight,and not more than about 100 μg/kg body weight. It will be understood byone of skill in the art that such guidelines will be adjusted for themolecular weight of the active agent. The dosage may also be varied forlocalized administration, e.g., for systemic administration, e.g., i.m.,i.p., i.v., and the like.

In another aspect, provided herein is a method of suppressingtumorigenicity of cells administered to a subject. As used herein, theterms “tumorigenicity” and “tumorigenic property” refer to the ability,tendency, or capability to cause, produce or develop tumors. The tumormay be benign (not cancerous), potentially malignant, pre-malignant(pre-cancerous), or malignant (cancerous). Examples of the benign,potentially malignant, or pre-malignant tumor used herein include,without limitation, adenoma, polyp, and teratoma. Suppressing orreducing tumorigenicity includes reducing or preventing one or moretumorigenic properties of cells. Preferably, a method of suppressingtumorigenicity comprises administering to the subject in need thereof acell composition comprising pluripotent stem cell-derived cells thathave been contacted to an effective amount of a compound thatselectively reduces or eliminates undifferentiated pluripotent stemcells or partially differentiated pluripotent stem cell derived progeny(e.g., a NAMPT inhibitor). Contacting to the compound can occur priorto, at the time of, or subsequent to administration of the cellcomposition to the subject to suppress tumorigenicity (e.g., potentialfor teratoma formation) of the administered cell composition. Forcontacting cells to the compound at the time of administration orsubsequent to administration of the cell composition to the subject, aneffective amount of the compound can be between about 0.1 to 0.126mg/m²/hour. Preferably, the subject is a human. Cells of the cellcomposition can be allogeneic or autogeneic to the subject. Preferably,suppressing or reducing tumorigenicity includes reducing, attenuating,or preventing one or more tumorigenic or potentially tumorigenicproperties of any cells within the cell composition. For example, amethod of the present invention is effective for inhibiting,suppressing, or reducing a tumorigenic property of a cell composition ifupon treatment of the cell population according to a method providedherein, the cell composition exhibits properties more similar to thoseproperties of (or those not found in) a cell composition comprisingnon-tumorigenic cells of the same species.

In a further aspect, the present invention provides methods forobtaining a cell composition having reduced tumorigenicity fortransplantation into a subject. In exemplary embodiments, the methodcomprises providing a population of pluripotent stem cell-derived cells;and contacting the population to an effective amount of a compound thatselectively reduces or eliminates undifferentiated pluripotent stemcells or partially differentiated pluripotent stem cell derived progeny(e.g., a NAMPT inhibitor), whereby the contacted population has reducedtumorigenicity relative to a pluripotent stem cell-derived cellpopulation not contacted to the compound, and using the contactedpopulation as a cell population for transplantation into the subject.Preferably, the subject is a human. Cells of the cell composition can beallogeneic or autogeneic to the subject.

Articles of Manufacture

In another embodiment of the invention, provided herein is an article ofmanufacture containing materials useful for methods of selectivelyreducing or eliminating undifferentiated pluripotent stem cells in vitroor in vivo. The article of manufacture comprises a container and alabel. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. In some cases, the container holds atherapeutic formulation provided herein and may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Theactive agent in the composition is a chemical or compound thatselectively reduces or eliminates undifferentiated pluripotent stemcells, or cocktail of two or more of such compounds. The label on, orassociated with, the container indicates that the composition is usedfor the in vitro and in vivo applications described herein. The articleof manufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

While the present invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described in detail. It shouldbe understood, however, that the description herein of specificembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, the intention is to cover allmodifications, equivalents and alternatives falling within the spiritand scope of the invention as defined by the appended claims.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1 Inhibition of an NAD⁺ Salvage Pathway ProvidesEfficient and Selective Toxicity to Human Pluripotent Stem Cells

Human embryonic stem cells (hESC) and induced pluripotent stem cells(hiPSC), collectively termed human pluripotent stem cells (hPSC), candifferentiate into almost any human cell type. Although significantprogress has been made in developing effective strategies for thedifferentiation of hPSC to progeny useful for drug toxicity testing andhuman disease modeling [1-4], continued safety issues preclude theirbroad use for human therapeutics. Cell heterogeneity, purity, and modeof transplantation are technically problematic, but the potentialformation of teratomas at the site of transplantation represents asignificant obstacle for clinical applications. Teratoma formation hasbeen reported in animal models following transplantation of low numbersof mouse and human PSC [5, 6] and after injection of hPSC-derived cells[7-10]. Pre-clinical testing of hESC-derived neural progenitor cells inthe Geron trial also resulted in cyst formation in the spines of mice,suggesting additional limitations for transplantation of hESC productsin human subjects [11]. Therefore, the elimination of potentiallytumorigenic pluripotent stem cells prior to transplantation is requiredbefore hPSC-based therapies can become widely available.

Here, we present data demonstrating that STF-31 (chemical structureshown in FIG. 9A) effectively eliminates these potentially tumorigeniccells across a range of culture conditions by a process that sparesdifferentiated progeny. These results provide an important advancementtowards the development of clinically safe hPSC-derived progeny forhuman stem cell based therapies.

STF-31 and WZB117 mediated toxicity in hPSC were directly compared toPluriSIn, a previously reported small molecule used for hPSCelimination. Treatment with STF-31 did not modify the morphology ofhiPSC following a 24 hour incubation (FIG. 1A). However, patches ofdiminished, non-adherent cells and reduction in overall cell number wereobserved following 48 hour treatment (FIG. 1A). After 72 hours ofexposure, STF-31 was highly toxic with a limited number of cellsremaining. In contrast to STF-31, it took 72 hours of treatment withWZB117 to cause morphological changes such as cell swelling, while therewere minimal alterations in the morphology of PluriSIn treated cells.These morphological changes are consistent with biochemical assays ofcell viability. STF-31 reduced cell viability to 10% following 72 hourstreatment as determined by the SYTOX® Green nucleic acid stain (FIG.1B). In contrast, there was no significant reduction in the viability ofPluriSIn or WZB117 treated cells. As the SYTOX® assay relies on thepresence of dead cells to quantify viability; we also performedlive/dead staining by calcein AM and ethidium homodimer-1 (FIG. 1C).Following 48 hours of STF-31 treatment, small patches of live cellsremained. These live cells were not visible by 72 hours of treatment. Incontrast, WZB117 and PluriSIn were not toxic to confluent hPSC. BecauseSTF-31 appeared to be the most robust small molecule for the eliminationof hPSC cultured under these conditions, we determined the median lethaldose (LD₅₀) of STF-31 for hiPSC to be 182 nM STF-31 48 hours aftercontinuous treatment (FIG. 1D). Increasing STF-31 concentrations to 100μM did not accelerate the time at which significant toxicity wasobserved by neutral red assay (FIG. 1D; FIG. 5A).

Because we were unable to fully reproduce the previously reported toxiceffects of PluriSIn on human pluripotent stem cells, we testedalternative culture conditions. Sub-confluent and confluent cells weregenerated either by plating cells at the same density and allowing themto grow for different lengths of time (24 vs 96 hours), or by platingthe cells at two different densities (1.5e5 vs 7.5e5 cells/cm²) andallowing them to grow for 24 hours (FIG. 1E). For each cell densitycondition, cells were treated with STF-31, PluriSIn, or WZB117 for 72hours. In these comparisons, the time required to detect STF-31 mediatedtoxicity was similar for both confluent and sub-confluent cells; withfewer than 2% viable cells remaining 72 hours after start of treatment.In contrast, toxicity of WZB117 and PluriSIn varied among the conditions(FIG. 1E). WZB117 was toxic to sub-confluent cells and confluent cellsthat were treated 24 hours post-plating, but was significantly lesstoxic to confluent cells that were treated 96 hours post-plating.PluriSIn was only effective on sub-confluent cells and did not inducesignificant toxicity in confluent cells. Moreover, increasingconcentrations of PlurSIn did not result in cell death in confluentcells (FIG. 6). Importantly, cell death in response to each of thecompounds was similar among three hESC (H1, H7, H9) (data not shown) andtwo hiPSC (KB3, DF6-9-9T) lines (FIG. 1; some data not shown), as wellas between hPSC cultured in either E8 or mTesR media compositions (FIG.6). Additionally, STF-31 was toxic to hESC colonies grown on humanfibroblast feeder cells with toxicity occurring between 48- 72 hours.Following 96 hours of treatment, toxicity was evaluated with brightfieldmicroscopy and staining for alkaline phosphatase activity. Colonies werenot detected with alkaline phosphatase staining; positive stainingconsisting of only non-adherent cells and acellular debris (FIG. 5A, toppanel). Brightfield microscopy confirmed an absence of colonies withnon-adherent cells with diminished morphology (FIG. 5A, middle panel).To confirm elimination of hESC, colonies were passaged 96 hours aftertreatment initiation and cultured for an additional 72 hours. No colonygrowth or alkaline phosphatase activity was detected in passaged cells,indicating elimination of hESC (FIG. 5A, lower panel).

Cell density is an important variable for many in vitro differentiationprotocols utilized to generate tissue-specific progeny from hPSC (e.g.,neuronal cells, hepatocytes, cardiomyocytes [19, 22, 24, 34]).Additionally, cell proliferation rates can also vary among common hPSCculture conditions and hPSC lines. To verify that STF-31 is toxic atvarious cell proliferation rates, hiPSC were incubated with5-bromo-2-deoxyuridine (BrdU) (10 μM) 24-96 hours post-plating and thecell cycle was examined (FIG. 1F; FIG. 5B). At 24 hours post-plating(low densities), 8% of cells are in G0/G1 phase, while at 96 hourspost-plating (high densities) 47% of the cells were in G0/G1 phase.Therefore, while all methods of elimination tested here were toxic torapidly proliferating hPSC, STF-31 was the only agent toxic to hPSCacross a broad range of hPSC densities and rates of proliferation. Thus,STF-31 mediated toxicity may be advantageous in confluent culturesystems where hPSC may have altered proliferation rates.

The effectiveness of STF-31 for the prevention of hPSC self-renewal andtumorigenicity was evaluated in vitro with a colony formation assaysalongside WZB117 and PluriSIn treatment. We determined that 24 hours wasthe minimal treatment time with STF-31 to achieve completetoxicity ofhPSC (data not shown). Using a 24 hour pulse treatment scheme, we foundthat STF-31 prevented the formation of alkaline phosphatase-positivecolonies from confluent hiPSC, whereas PluriSIn and WZB117 did notprevent colony growth (FIG. 1G). As a more stringent examination of theeffectiveness of STF-31 for the removal of hiPSC, a colony formationassay was performed without cell passaging to eliminate exaggerated celldeath caused by stress during the dissociation and re-plating of cells(FIG. 1H). In this strategy, STF-31, WZB117, and PluriSIn were appliedfor 24 hours to confluent hiPSC then media was replaced daily for sixdays. Similar levels of alkaline phosphatase-positive cells wereobserved in the DMSO, WZB117, and PluriSIn-treated hiPSC. In contrast, alimited number of colonies formed in hiPSC treated with 2.5 μM STF-31and no colonies were observed in the hiPSC treated with 5 μM STF-31.These findings demonstrate that for confluent hiPSC, 24 hours treatmentwith STF-31 prevents reformation of alkaline phosphatase-positivecolonies, while PluriSIn and WZB117 are ineffective against a confluentmonolayer of hPSC.

To further evaluate the utility of STF-31, we examined the effects of a24 hour pulse treatment of STF-31 treatment on sub-confluent andconfluent hiPSC as well as differentiated cells. The LD₅₀ of STF-31 wasdetermined to be 1.35 μM and 1.78 μM when sub-confluent or confluentcells were treated, respectively (FIG. 2A). Consistent with the data inFIG. 1, 24 hour treatment with WZB117 and PluriSIn eliminatedsub-confluent hiPSC, but not confluent hiPSC (FIG. 2B). 24 hourtreatment with STF-31 did not appear to be toxic to human fibroblasts,as concentrations of 100 μM only reduced fibroblast viability by 30%.These data for the 24 hour treatment are consistent with our previousreport that exposed fibroblasts to STF-31 for 72 hours continuously[19]. Using this 24 hour pulse treatment, the LD₅₀ of STF-31 onhiPSC-derived cardiomyocyte cultures was 40 μM, a 22-fold higherconcentration than required for hiPSC (FIG. 2A). Extending the treatmenttime to 72 hours did not result in a decrease in cell viability withfully committed retinal pigmented epithelial cells (ARPE-19 line; datanot shown) or fibroblasts [19].

Using the conditions established for hPSC elimination, (5 μM for 24hours) the effects of STF-31 on day 10 cardiomyocyte function andmarkers were interrogated. STF-31 treatment did not affect cardiomyocytespontaneous contractility (data not shown). A pulse treatment withSTF-31 did not affect abundance of cardiac markers TNNI3 and IRX4 asmeasured by flow cytometry (FIG. 2C) and treated cardiomyocytesmaintained comparable structural organization of TNNT2, which is foundin cardiac sarcomeres, (FIG. 2D) 72 hours after treatment initiation.Expression of cardiac genes as measured by q-PCR was not significantlyaltered after initiation of STF-31 treatment (FIG. 2E).

To confirm selective toxicity in the presence of differentiated cells,colony formation assays were performed with titrating amounts of hiPSCwith either day 10 cardiomyocytes (FIG. 2F) or human fibroblasts (FIG.2G). Initially, a 24-hour pulse treatment was used and resulted inelimination of a majority of hiPSC colonies from day 10 cardiomyocytes,however some colonies remained after treatment (FIG. 5C). A 48 hourpulse treatment with 5 μM STF-31 was found to completely eliminatehiPSC. Both the fibroblasts and cardiomyocytes remained after treatmentand spontaneous contraction was observed in the cardiomyocytes at thetime of alkaline phosphatase staining (data not shown) and up to 10 daysafter treatment initiation (data not shown). Altogether, these findingsdemonstrate that a pulse treatment with STF-31 is an effective strategyfor selective elimination of remnant hPSC across a range of progeny.

In cancer cells, STF-31 was initially described as a GLUT1 inhibitorthat induces necrosis [35]. However, because of the differences intiming and efficacy that we observe between STF-31 and WZB117 (anirreversible GLUT1 inhibitor), we set out to further explore themechanism of action of STF-31 in hPSC. In these studies, glucosedeprivation was used for comparison as it has been previously reportedas a method for hPSC elimination [16]. Similar to the results observedfor WZB117, glucose deprivation was toxic to sub-confluent cells, buttoxicity was both delayed and decreased in confluent cells (FIG. 3A).Using extracellular flux analysis, the extracellular acidification rate(ECAR) was measured to provide information on glycolysis and oxygenconsumption rate (OCR) was monitored to probe mitochondrial respiration.Treatment of hiPSC with the GLUT1 inhibitor WZB117 led to a decrease inECAR by the earliest timepoint measured (FIG. 3B), acting in a mannersimilar to that observed with 2-deoxyglucose (2-DG), an inhibitor ofglucose metabolism (FIG. 7A). In contrast, the decrease in ECARfollowing STF-31 treatment was significantly delayed by up to 15 hoursas compared to the inhibitors of glucose oxidation (FIG. 3B). Resultsfor OCR are similar to ECAR, as hiPSC treated with STF-31 required 15 hbefore reductions in OCR were observed. This finding is in contrast tothe effects of WZB117, which decreased OCR by the first time pointexamined (8 hours) (FIG. 3B).

Consistent with the effects on glucose metabolism, STF-31 did not alterglucose uptake in hiPSC following 1 hour of treatment, while 1 hourtreatment with WZB117 resulted in a 20% decrease in uptake (FIG. 3C).The first time point at which STF-31 caused a decrease in glucose uptakewas 18 hours after treatment, which was 3 hours after the decrease inECAR. Thus, the significant decrease in glucose uptake that isconsistent with results previously reported in cancer [35] occurredafter STF-31-mediated inhibition of glycolysis and mitochondrialoxidative metabolism. The delayed effects of STF-31 on glucose uptakewere unexpected for an inhibitor affecting either glucose transport orGLUT1 biogenesis and are not consistent with the reported mechanism ofaction for STF-31 as a GLUT1 inhibitor.

WZB117 and STF-31 have been reported to induce necrosis in cancer cellsbased on annexin staining at 24-48 hours and 24-72 hours of treatment,respectively, but other reports have associated a decrease in ATP due toglucose starvation with apoptosis [36-38]. To determine how WZB117 andSTF-31 promote hiPSC death, effects on energy sensing pathways wereexamined by measuring phosphorylation of AMP-activated protein kinase(p-AMPK) in sub-confluent cells. In response to WZB117 treatment andglucose deprivation, p-AMPK was detected as early as 12 hourspost-treatment and total levels of AMPK decreased by 24 hours followingWZB117 treatment of hiPSC (FIG. 7B). In response to STF-31 treatment,there was an increase in p-AMPK at 24 hours, approximately 8 hours afterthe decrease in ECAR and OCR. The timing of AMPK activation proveddistinct between STF-31 and both glucose deprivation and WZB117treatment. Moreover, in WZB117-treated and glucose-deprived cells,cleaved caspase-3 and -9 were observed at 12 hours and remained elevateduntil 36 hours, at which time a decrease in cleaved caspase-3 and -9levels are observed (FIG. 3D). Induction of caspase-3/7 activity wasalso observed as early as at 12 hours (FIG. 7C). In contrast, STF-31caused caspase-3 and -9 cleavage significantly later (FIG. 3D) and thiscoincided with caspase-3/7 activity at 24 hours after initiation oftreatment (FIG. 7C). Thus, WZB117 and STF-31 appear to act on hiPSCthough activation of caspase-mediated cell death pathways.

The temporal discordance among metabolism, glucose uptake, and caspaseactivation (FIG. 4) prompted us to measure adenine and pyridinenucleotide levels because they are known to affect both glycolytic andoxidative metabolism and cell viability [33]. These analyses revealedthat with STF-31 treatment, NAD⁺ levels are reduced to ˜50% by 3 hoursand to <1% by 18 hours. ATP levels do not decrease until 18 hours (FIG.3E). These data suggest that STF-31 leads to an inhibition of the NAD⁺de novo synthesis or salvage pathways. To investigate which NAD⁺ pathwayis being targeted, rescue experiments were performed using metabolitesadded in the presence of STF-31. The addition of 1 and 10 μM nicotinicacid restored hPSC viability to 78% and 97%, respectively (FIG. 3F).Nicotinic acid is normally not present in hPSC culturing conditions andcan be utilized by nicotinic acid phosphoribosyltransferase (NAPRT) inone of the thee NAD⁺ salvage pathways. Alternatively, the addition of 10and 100 μM nicotinamide, the substrate for nicotinamidephosphoribosyltransferase (NAMPT), failed to rescue STF-31 mediatedtoxicity (data not shown). Altogether, these data suggest that STF-31inhibits the NAMPT dependent NAD⁺ salvage pathway.

To assess STF-31′s toxicity to hiPSC-derived progeny and terminallydifferentiated cells, we cultured mesenchymal stem cells, fibroblasts,retinal pigmented epithelial cells, hiPSC-derived cardiomyocytes,specified hepatic endoderm cells, and neural progenitors in the presenceof STF-31. None of these cell types exhibited visible changes inmorphology or obvious cell death with STF-31 treatment (FIG. 8A).Viability measurements were consistent with these visual observations.See data for cardiomyocytes, neural progenitors, and fibroblasts inFIGS. 8B-C). Importantly, cardiomyocytes treated with STF-31 on day 5(progenitor) and day 10 (committed; not terminally differentiated) ofdifferentiation exhibit spontaneous rhythmic contractions, protein andmRNA levels of cardiomyocyte markers, and structural featuresindistinguishable from controls (data for cells treated on day 10 inFIG. 8B). See Boheler et al., Stem Cell Reports 3, 185-203 (2014).Altogether, these findings suggest STF-31 is suitable for a wide rangeof hPSC derivatives.

This study establishes and defines an effective strategy for selectivelytoxicity of hPSC. Pluripotent stem cell metabolism is characterized byaerobic glycolysis, decreased mitochondrial membrane potential, andincreased reliance on anabolic processes [39]. Enhanced expression ofGLUT1 and reliance on glycolytic metabolism make glucose deprivation[16] or inhibition of GLUT1 promising strategies for selectiveelimination of hPSC as we previously described. See Boheler et al., StemCell Reports 3, 185-203 (2014). However, in our culture system, we foundthat glucose deprivation, WZB117, and PluriSIn had limited to notoxicity on confluent monolayers of hPSC, an observation that wasconsistent among five hPSC lines and two different media compositions.Therefore, these strategies may not be ideal for applications thatrequire confluent cell monolayers or altered proliferation rates derivedfrom hPSC, such as cardiomyocytes, hepatocytes, and neuronal cultures[19, 22, 24, 34].

We show that hPSC can be selectively targeted using STF-31, which offersideal toxicity characteristics for culture systems that requireconfluent monolayers of cells for differentiation or produce confluentmonolayers of differentiated cells. Regenerative medicine strategiesthat require tissues, scaffolds, or other thee-dimensional cell productsshould therefore benefit from these findings. STF-31 offers advantagesover current approaches as it exhibits continued toxicity after a 24 or48 hours pulse treatment. This toxicity is independent of cell densityand provides for a 22-fold difference between the LD₅₀ of confluent hPSCand hPSC-derived cardiomyocytes. Moreover, treatment of a co-culturewith cardiomyocytes and hiPSC provided selective elimination of thehPSC, while sparing cardiomyocytes. Finally, 5 μM STF-31 was sufficientto prevent hPSC colony self-renewal and regrowth for 7 days in vitro;however, the traditional colony formation assay that involvesdissociation and re-plating of hPSC indicates lower concentrations ofSTF-31 may also be effective.

STF-31 has been reported to inhibit GLUT1 in cancer cells [35] and iscurrently marketed as a GLUT1 inhibitor. However, our data forSTF-31-mediated toxicity and effects on metabolic flux do not supportthis mechanism of action. Rather, these data show that the effects onglycolytic metabolism are due to depletion of NAD⁺. STF-31-mediatedtemporal effects on metabolism (glycolysis and oxidativephosphorylation) differ from both glucose deprivation and WZB117.Additionally, the failure to block glucose uptake prior to inhibition ofglycolytic flux demonstrates that STF-31-mediated toxicity in hPSC isnot due to GLUT1 inhibition. As a result of these findings, we set outto define the mechanism of action of STF-31. Our data reveal that STF-31targets NAMPT, the enzyme that catalyzes the rate-limiting step in theconversion of nicotinamide to NAD⁺ in one of the NAD⁺ salvage pathways.Further support of this mechanism is found in a separate study that waspublished while our manuscript was being prepared. Dragovich et al.systematically synthesized 67 different 3-aminopyridine-derived amidesand screened them for their ability to inhibit NAMPT [40]. From thisscreen, Compound 51, which has the same chemical structure as STF-31,was found to directly inhibit NAMPT in a purified enzyme assay (IC₅₀=19nM). By X-ray crystallography, STF-31 was shown to bind in the ligandbinding pocket of NAMPT. Altogether, these data confirm that STF-31mediated toxicity is caused by inhibition of an NAD⁺ salvage pathway bytargeting NAMPT. Additionally, our study provides evidence that thetoxic effects mediated by STF-31 in hPSC can be attributed to depletionof NAD⁺, not inhibition of glucose transport. This discovery providesfundamental insight into the basic metabolic profile of pluripotency, asthese cells are less able to compensate for a loss of NAD⁺ salvagepathways than their differentiated progeny. The reliance on NAD⁺ salvagepathways shown here is consistent with a recent report that usedcompound FK866 to inhibit NAMPT and showed that adequate NAD⁺ levels arerequired to establish and maintain pluripotency during reprogramming[41]. However, the report by Son et al., did not demonstrate completecell death of hPSC in the conditions tested. Going forward, more potentand water-soluble inhibitors of NAMPT should be important reagents forthe preparation of clinically relevant cells and tissues derived fromhPSC.

In summary, this study establishes that targeting the NAD⁺ salvagepathway mediated by NAMPT represents an effective and efficient strategyfor selective hPSC toxicity. Our detailed analyses of the cellularmetabolic events further support the use of NAMPT inhibitors overglucose starvation or GLUT1 inhibition for the elimination of hPSC inculture. When using STF-31 to inhibit NAMPT in a 24 or 48 hours pulsetreatment scheme, hPSC elimination can be achieved across many cultureconditions without cytotoxic effects on the terminally differentiatedcells and hPSC-derived progeny tested in this study.

Materials and Experimental Procedures:

Cell Culture and Reagents: hiPSC (DF6-9-9T, KB3 [19, 20]) and hESC (H1,H7, H9 [21]) were cultivated in monolayer culture on Matrigel in E8 ormTeSR medium as previously described [6, 19]. For all experiments, hPSCwere plated at 1.5e5 total cells per 2 cm², unless otherwise specified,as plating an explicit number of cells during routine passaging enhancesreproducibility among experiments and maintains high quality monolayercultures of pluripotent cells. Treatment with small molecule inhibitorswas initiated between 24 and 96 hours post-plating. These time pointsrepresent the time at which cells are typically 15-20% and 100%confluent, respectively. hiPSC derived cardiomyocytes weredifferentiated and maintained as previously described[22]. For hESCcolonies that were grown on a feeder cell layer, H1 colonies werecultured on mitotically inactivated human fibroblast feeders in E8media, colonies were passaged with collagenase IV for 40 min at 37° C.[23]. PAX6-positive neuronal progenitors were differentiated asdescribed [24]. Human fibroblasts were cultured as previously described[19]. Small molecules STF-31(4-[[[[4-(1,1-Dimethylethyl)phenyl]sulfonyl]amino]methyl]-N-3-pyridinylbenzamide,Tocris), WZB117 (EMD Millipore), PluriSIn (Sigma-Aldrich), nicotinicacid (Sigma-Aldrich), and nicotinamide (Sigma-Aldrich) were used totreat cells. For glucose deprivation, E8 media was prepared as described[19] with the modification that DMEM F-12 without glucose (USBiological) was used. For glucose deprivation media control, 17.5 mMglucose (Sigma-Aldrich) was added into glucose free E8 media.

In Vitro Toxicity Assays: Treatment with small molecules or glucosedeprivation was initiated in hESC or hiPSC at 24 and 96 h post-platingand in vitro toxicity assays were performed at specified treatmentendpoints 24-96 hours after treatment initiation. For pulsed treatment,hiPSC were treated with STF-31 for 24 hours, washed twice with D-PBS,and cultured in media for an additional 48 hours. Neutral red assays forcell viability were performed as previously described [25]. In vitrocell death was determined using SYTOX® Green nucleic acid stain (LifeTechnologies). Briefly, cells were incubated in 5 μM SYTOX® Green for 30minutes at 37° C. in a humidified cell incubator with 5% CO₂. Percentcell viability was determined by normalizing to replicate cellsincubated with 120 μM digitonin (Sigma-Aldrich) and 5 μM SYTOX® Green.Imaging of cell viability was performed using Live/Dead®Viability/Cytotoxicity Kit, for mammalian cells (Life Technologies).Cells were stained for 20 minutes at room temperature with 4 μM CalceinAM to detect viable cells and 2 μM Ethidium Homodimer 1 to detect cellswith compromised membranes. Representative images of alterations in cellmorphology were acquired on confluent hPSC 24-72 hours after treatmentinitiation. Imaging was performed with a Nikon Ti-U inverted microscope.

BrdU Incorporation and Flow Cytometry: Cells were plated at 7.5e5 cellsper 9.6 cm² and 10 μM 5-bromo-2-deoxyuridine (BrdU) was incorporated inhiPSC 24-96 h post-plating for 1 hour at 37° C. in a humidified cellincubator with 5% CO₂. After incorporation, cells were collected andstained using FITC BrdU Flow Kit per manufacturer's guidelines (BI)Biosciences). Cell viability was determined using Fixable Viability DyeeFluor® 450 (eBiosciences). Analyses were performed on 30,000 eventsacquired on a BD LSRII flow cytometer (BD Biosciences), using FCSExpressV3 (DeNovo Software). The percent of cells in each phase of the cellcycle was determined by gating on populations within each phase in thelive cell population.

Colony Formation Assay: The colony formation assay was performed inthree different variations on confluent hiPSC that were treated with 2.5and 5 μM STF-31, 30 μM WZB117, 20 μM PluriSIn for 24 hours. The firstiteration was adapted from previously described method [26], after 24hours of treatment, hiPSC were detached with Accutase (StemCellTechnologies), re-suspended in E8, passed though 12×75 mm tube with cellstrainer cap (Falcon), and plated at 5e4 live cells per 2 cm². Mediawere changed every 48 hours and cells were cultured for six days, atwhich time staining for alkaline phosphatase was performed withleukocyte alkaline phosphatase kit (Sigma-Aldrich) as described [27] andplates were visually inspected for phosphatase-positive colonies. Thisstrategy represents the most common implementation of the colonyformation assay. However, concerns regarding the cell death normallyencountered during passaging prompted another approach for stricterassessment. In the second iteration, cells were treated for 24 hours,washed twice with 0.5 mL D-PBS, and media refreshed with E8. Thereafter,media were replaced daily for six days at which point staining foralkaline phosphatase activity was performed. In this version, cells donot undergo the stress of passaging, which may result in erroneously lowviability that is not a direct result of the compound; thus, thismodified assay is considered a more stringent assessment of hPSCelimination than the former. Elimination of hiPSC from co-cultures withdifferentiated cells was performed with human fibroblasts and day 10cardiomyocytes derived from hiPSC. hiPSC (DF6-9-9T) were plated atconcentrations ranging from 100-10,000 live cells per well with humanfibroblasts (1.75e5 cells) or day 10 differentiated cardiomyocytes(3.75e5 cells) in E8 and Y-27632 per 9.6 cm². After 24 hours, treatmentwas performed with 5 μM STF-31 for 24 or 48 hours, washed twice andmedia refreshed daily with E8. Six days after plating, staining foralkaline phosphatase activity was performed. For elimination of hESCcolonies grown on mitotically inactivated human fibroblast feeders,colonies were treated for 96 hours with 2.5 μM STF-31 at which pointcells were imaged with brightfield microscopy and stained for alkalinephosphatase activity or colonies were passaged with collagenase IV (2mg/mL for 40 minutes at 37° C.) and cultured for an additional 72 hoursand stained for alkaline phosphatase activity. Imaging of wells wasperformed with a Sony Cyber-shot DSC-TX30 and individual colonies with aNikon stereoscope.

Characterization of Cardiomyocytes: For all experiments, a 24-hour pulsetreatment with 5 μM STF-31 was performed on day 10 cardiomyocytedifferentiation cultures derived from hiPSC (DF6-9-9T). Flow cytometrywas performed as previously described 72 hours after initiation ofSTF-31 treatment for Troponin I type 3 (TNNI3) and Iroquois-classhomeodomain protein (IRX4) [19]. Quantitative-real time PCR for TNNI3,Troponin T2 (TNNT2), and Homeobox protein Nkx-2.5 (NKX2-5) was performedas previously described using Taqman® Assays [19] (Life Technologies)24-72 hours after initiation of STF-31 treatment. For immunoflourescentdetection of TNNT2, cardiomyocytes were passaged onto coverslips 72hafter initiation of STF-31 treatment and cultured for an additional 48hours. Cells were then fixed with 4% paraformaldehyde, permeabilizedwith 0.2% Triton-X, incubated with primary antibody for Troponin T(TNNT2) (Abcam: ab8295) overnight at 4° C. Cells were then incubated insecondary antibody goat anti-mouse IgG1-Alexa 568 (Life Tech: A-21124)and nuclei were detected with Hoechst 33342 (Life Tech: H21492). Cellswere imaged with a Nikon Ti-U inverted microscope and Nikon Eclipse 90iconfocal microscope.

Extracellular Flux Analysis: hiPSC were plated at 6e4 cells per well onspecialized microplate (Seahorse Bioscience) as previously described[28] with several exceptions. Extracellular flux analysis was performedon hiPSC 48 hours post-plating using Seahorse Bioscience XF24 Analyzer.Cells were treated with 2.5 μM STF-31, 30 μM WZB117, 20 mM2-deoxyglucose (2-DG; Sigma-Aldrich), vehicle control, or media controlfor 5.5 h, washed twice with 750 μL assay medium, and placed in 750 μLassay medium containing the appropriate treatment. The microplate wasequilibrated in non-CO₂ incubator for 1.5 h, and analyzed for 16 h inSeahorse Bioscience XF24 Analyzer. Assay medium consists of basal E8reagents with the following exceptions: basal DMEM F-12 without phenolred (Gibco), no sodium bicarbonate, and 2.5 mM GlutaMAX™ (Gibco) and 15mM Hepes (Gibco). Assay medium was adjusted to pH 7.4 at 37° C. prior touse. Extracellular acidification rate (ECAR) and basal oxygenconsumption rate (OCR) were collected approximately every 60 minutes.Treatments were normalized to baseline media control value and arerepresented as average of thee biological replicates.

Glucose Uptake Assay: Sub-confluent hiPSC ( 24 hours post-plating) weretreated with 2.5 μM STF-31, 30 μM WZB117, 20 μM Cytochalasin B(Sigma-Aldrich), and vehicle control for 1, 15, 18, and 24 hours. For 1hour treatment, cells were placed in Krebs-Ringer Hepes (KRH) containingtreatment at 37° C. in a humidified cell incubator with 5% CO₂. For 15,18, and 24 hour treatments, cells were treated in E8 medium for 14, 17,and 23 hours, respectively, at which point media was changed to KRHbuffer containing specific treatment for 1 hour. After specifiedtreatment time, glucose uptake was performed with 0.5 μCi [³H]2-deoxyglucose for 5 min at 37° C. as previously described [29]. Uptakedata were normalized to protein concentration measured with Lowrymethod.

Immunoblot Analysis: Non-adherent cells were collected bycentrifugation, adherent cells were washed once with d-PBS and lysed inLaemmli buffer, combined with non-adherent cell pellet, and heated at95° C. for 5 minutes. Protein concentration was measured using the Qubitprotein assay (Life Technologies). 25 μg of total protein was separatedby SDS-PAGE, transferred to nitrocellulose membrane (GE Healthcare LifeSciences) and blocked according to the manufacturer's instructions.Membranes were incubated overnight at 4° C. with the following dilutionsof primary antibodies: rabbit anti-cleaved caspase-3 (1:500), rabbitanti-caspase-9 (1:1000), rabbit anti-phospho-AMPK (1:1000), and rabbitanti-AMPK (1:1,000) from Cell Signaling Technology, and mouse anti-GAPDH(1:10,000) from Life Technologies. Membranes were then incubated withsecondary antibodies for 45 minutes at the following concentrations:donkey anti-mouse-horseradish peroxidase (1:5,000) and donkeyanti-rabbit-horseradish peroxidase (1:7,000) from Jackson ImmunoresearchLaboratories, Inc., followed by detection using enhancedchemiluminescence [30].

Cleaved caspase-3/7 fluorescence based assay: Treatment with 2.5 μMSTF-31, 30 μM WZB117, or glucose deprivation was initiated insub-confluent hiPSC ( 24 hours post-plating) for 6, 12, 18, and 24 hoursat which time cleaved caspase-3/7 activity was measured as previouslydescribed [31, 32] with the following exceptions: cells were cultured in500 μL E8 media and 250 μL of 3× caspase buffer was added to media atendpoint.

Nucleotide Pool Measurements: ATP, ADP, AMP, and NAD⁺ were extractedusing perchloric acid precipitation and analyzed using HPLC, following apreviously published method [33]. ATP, ADP, AMP and NAD⁺ peaks weremeasured for each sample, compared with the standards, and normalized toprotein levels.

Statistical Analysis: All experiments were performed in a minimum ofthree biological replicates. Data are represented as mean with standarderror of the mean for N biological replicates. Statistical analysis wasperformed using one-way ANOVA with Tukey post hoc test.

REFERENCES

-   1. Chong, J. J., et al., Human embryonic-stem-cell-derived    cardiomyocytes regenerate non-human primate hearts. Nature, 2014.-   2. Ebert, A. D., P. Liang, and J. C. Wu, Induced pluripotent stem    cells as a disease modeling and drug screening platform. J    Cardiovasc Pharmacol, 2012. 60(4): p. 408-16.-   3. Grskovic, M., et al., Induced pluripotent stem    cells—opportunities for disease modelling and drug discovery. Nat    Rev Drug Discov, 2011. 10(12): p. 915-29.-   4. Takahashi, K. and S. Yamanaka, Induced pluripotent stem cells in    medicine and biology. Development, 2013. 140(12): p. 2457-61.-   5. Hentze, H., et al., Teratoma formation by human embryonic stem    cells: evaluation of essential parameters for future safety studies.    Stem Cell Res, 2009. 2(3): p. 198-210.-   6. Lawrenz, B., et al., Highly sensitive biosafety model for    stem-cell-derived grafts. Cytotherapy, 2004. 6(3): p. 212-22.-   7. Cui, L., et al., WNT signaling determines tumorigenicity and    function of ESC-derived retinal progenitors. J Clin Invest, 2013.    123(4): p. 1647-61.-   8. Doi, D., et al., Prolonged maturation culture favors a reduction    in the tumorigenicity and the dopaminergic function of human    ESC-derived neural cells in a primate model of Parkinson's disease.    Stem Cells, 2012. 30(5): p. 935-45.-   9. Kroon, E., et al., Pancreatic endoderm derived from human    embryonic stem cells generates glucose-responsive insulin-secreting    cells in vivo. Nat Biotechnol, 2008. 26(4): p. 443-52.-   10. Lee, A. S., et al., Tumorigenicity as a clinical hurdle for    pluripotent stem cell therapies. Nat Med, 2013. 19(8): p. 998-b    1004.-   11. DeFrancesco, L., Fits and starts for Geron. Nat    Biotechnol, 2009. 27(877).-   12. Cao, F., et al., Molecular imaging of embryonic stem cell    misbehavior and suicide gene ablation. Cloning Stem Cells, 2007.    9(1): p. 107-17.-   13. Rong, Z., et al., A scalable approach to prevent teratoma    formation of human embryonic stem cells. J Biol Chem, 2012.    287(39): p. 32338-45.-   14. Ben-David, U., N. Nudel, and N. Benvenisty, Immunologic and    chemical targeting of the tight-junction protein Claudin-6    eliminates tumorigenic human pluripotent stem cells. Nat    Commun, 2013. 4: p. 1992.-   15. Tang, C., et al., An antibody against SSEA-5 glycan on human    pluripotent stem cells enables removal of teratoma-forming cells.    Nat Biotechnol, 2011. 29(9): p. 829-34.-   16. Tohyama, S., et al., Distinct metabolic flow enables large-scale    purification of mouse and human pluripotent stem cell-derived    cardiomyocytes. Cell Stem Cell, 2013. 12(1): p. 127-37.-   17. Ben-David, U. and N. Benvenisty, Chemical ablation of    tumor-initiating human pluripotent stem cells. Nat Protoc, 2014.    9(3): p. 729- 40.-   18. Ben-David, U., et al., Selective elimination of human    pluripotent stem cells by an oleate synthesis inhibitor discovered    in a high-throughput screen. Cell Stem Cell, 2013. 12(2): p. 167-79.-   19. Boheler, K. R., et al., A human pluripotent stem cell surface    N-glycoproteome resource reveals markers, extracellular epitopes,    and drug targets. Stem Cell Reports, 2014. 3(1): p. 185-203.-   20. Yu, J., et al., Human induced pluripotent stem cells free of    vector and transgene sequences. Science, 2009. 324(5928): p.    797-801.-   21. Thomson, J. A., et al., Embryonic stem cell lines derived from    human blastocysts. Science, 1998. 282(5391): p. 1145-7.-   22. Bhattacharya, S., et al., High efficiency differentiation of    human pluripotent stem cells to cardiomyocytes and characterization    by flow cytometry. J Vis Exp, 2014(91): p. 52010.-   23. Amit, M. and J. Itskovitz-Eldor, Morphology of Human Embryonic    and Induced Pluripotent Stem Cell Colonies Cultured with Feeders, in    Atlas of Human Pluripotent Stem Cells, M. Amit and J.    Itskovitz-Eldor, Editors. 2012, Humana Press. p. 15-39.-   24. Shi, Y., et al., Human cerebral cortex development from    pluripotent stem cells to functional excitatory synapses. Nat    Neurosci, 2012. 15(3): p. 477-86, 51.-   25. Repetto, G., A. del Peso, and J. L. Zurita, Neutral red uptake    assay for the estimation of cell viability/cytotoxicity. Nat    Protoc, 2008. 3(7): p. 1125-31.-   26. O'Connor, M. D., M. D. Kardel, and C. J. Eaves, Functional    assays for human embryonic stem cell pluripotency. Methods Mol    Biol, 2011. 690: p. 67-80.-   27. Rao, S., et al., Differential roles of Sall 4 isoforms in    embryonic stem cell pluripotency. Mol Cell Biol, 2010. 30(22): p.    5364-80.-   28. Zhang, J., et al., Measuring energy metabolism in cultured    cells, including human pluripotent stem cells and differentiated    cells. Nat Protoc, 2012. 7(6): p. 1068-85.-   29. Yamamoto, N., et al., Measurement of glucose uptake in cultured    cells. Curr Protoc Pharmacol, 2011. Chapter 12: p. Unit 12 14 1-22.-   30. Khan, P., et al., Luminol-Based Chemiluminescent Signals:    Clinical and Non-clinical Application and Future Uses. Appl Biochem    Biotechnol, 2014. 173(2): p. 333-355.-   31. Meares, G. P., et al., AMP-activated protein kinase attenuates    nitric oxide-induced beta-cell death. J Biol Chem, 2010. 285(5): p.    3191-200.-   32. Carrasco, R. A., N. B. Stamm, and B. K. Patel, One-step cellular    caspase-3/7 assay. Biotechniques, 2003. 34(5): p. 1064-7.-   33. Broniowska, K. A., et al., Effect of nitric oxide on    naphthoquinone toxicity in endothelial cells: role of bioenergetic    dysfunction and poly (ADP-ribose) polymerase activation.    Biochemistry, 2013. 52(25): p. 4364-72.-   34. Mallanna, S. K. and S. A. Duncan, Differentiation of hepatocytes    from pluripotent stem cells. Curr Protoc Stem Cell Biol, 2013.    26: p. Unit 1G4.-   35. Chan, D. A., et al., Targeting GLUT1 and the Warburg effect in    renal cell carcinoma by chemical synthetic lethality. Sci Transl    Med, 2011. 3(94): p. 94ra70.-   36. Altman, B. J. and J. C. Rathmell, Metabolic stress in autophagy    and cell death pathways. Cold Spring Harb Perspect Biol, 2012.    4(9): p. a008763.-   37. Coloff, J. L., et al., Akt requires glucose metabolism to    suppress puma expression and prevent apoptosis of leukemic T cells.    J Biol Chem, 2011. 286(7): p. 5921-33.-   38. Zhao, Y., et al., Glucose metabolism attenuates p53 and    Puma-dependent cell death upon growth factor deprivation. J Biol    Chem, 2008. 283(52): p. 36344-53.-   39. Folmes, C. D., et al., Energy metabolism plasticity enables    sternness programs. Ann N.Y. Acad Sci, 2012. 1254: p. 82-9.-   40. Dragovich, P. S., et al., Fragment-based design of    3-aminopyridine-derived amides as potent inhibitors of human    nicotinamide phosphoribosyltransferase (NAMPT). Bioorg Med Chem    Lett, 2014. 24(3): p. 954-62.-   41. Son, M. J., et al., Nicotinamide overcomes pluripotency deficits    and reprogramming barriers. Stem Cells, 2013. 31(6): p. 1121-35.

Example 2 FK866 Toxicity to Human Pluripotent Stem Cells

FIG. 10 presents cell viability data for hiPSCs treated with FK866 (FIG.9B) for up to 72 hours. Timing of pluripotent stem cell death for thisNAMPT inhibitor was similar to that observed for STF-31. It was alsoobserved that FK866 was toxic to human pluripotent stem cells at lowerconcentrations than STF-31. These data suggest that STF-31 and FK866will be clinically relevant and are consistent with our findings thatSTF-31 does not affect differentiated progeny in culture. Therefore, aneffective strategy for selectively eliminating pluripotent stem cells isto deplete NAD⁺ levels using a NAMPT inhibitor. Moreover, these datademonstrate that NAMPT inhibition provides a rapid, scalable, andinexpensive method that works independent of media composition, celldensity, and cell line—making the strategy more universally applicablethan those previously reported. Importantly, as STF-31 and FK866 canreduce cancer mass in vivo, these studies suggest their anti-tumorproperties are not limited to pluripotent cells. Thus, these compoundswill be useful for selectively eliminating tumorigenic cells in cellsprepared for transplantation, independent of whether teratoma-causingcells in differentiated cultures are pluripotent or comprise one or moredifferentiated phenotypes.

Example 3 STF-31 Spares Human Pluripotent Stem Cell Derived Progeny andTerminally Differentiated Cells

Mesenchymal stem cells, fibroblasts, retinal pigmented epithelial cells,human pluripotent stem cell-derived cardiomyocytes, specified hepaticendoderm cells, and neural progenitors show no visible changes inmorphology or obvious cell death with STF-31 treatment (FIG. 8A) andviability measurements are consistent with these observations (FIGS. 5A,5B). Human pluripotent stem cell derived cardiomyocytes and neuralprogenitor cells treated with STF-31 demonstrate protein and mRNA levelsof markers and structural features indistinguishable from controls(FIGS. 8B-C). As shown in FIG. 11, adult pigmented retinal epithelialcells (ARPE) and PAX6 positive neural progenitor cells are alsoresistant to STF-31 treatment. It was observed that the cell deathobserved in neural progenitor cells treated for 72 hours continuouslywith STF-31 can be rescued with addition of exogenous NA, consistentwith the mechanism of NAMPT inhibition observed in the pluripotent stemcells. Altogether, these data demonstrate that STF-31 is suitable for awide range of hPSC derivatives and that short term treatment ( 24 hours)of the neural progenitors and ARPE cells does not result in significanttoxicity.

Example 4 Elimination of Tumorigenic Pluripotent Stem Cell-DerivedProgeny in Vitro and in Vivo

It is possible that tumors may form from cells that no longer fit theclassical functional definition of a pluripotent stem cell. For example,partially differentiated or progenitor cells may self-renew and betumorigenic, but may not give rise to cells from all three germ layers(thus, precluding them from fitting the functional definition of a truepluripotent cell). In this example, pluripotent stem cell-derivedtumorigenic progeny include the following: immature progenitor cells,partially differentiated progeny, fetal-like progeny, non-terminallydifferentiated progeny, as well as progeny that retain or reactivatepluripotent tumorigenic regulatory pathways and/or tumorigeniccharacteristics through genetic instability or activation of oncogenicpathways. Here, the compound is used to eliminate neoplasia's initiatedfrom pluripotent stem cell-derived tumorigenic progeny in conjunctionwith administration of pluripotent stem cell-derived progeny to themammal (e.g. transplantation of human pluripotent stem cell-derivedcardiomyocytes into human). The compound is administered at the sametime that the cells or tissue product are delivered, or at a timepost-cell delivery (e.g., 1 day to 30 days post-cell injection). Thecompound may include STF31, FK866, other NAMPT inhibitor, or acombination of NAMPT inhibitors. For example, the compound isadministered as a 96 hour continuous infusion every 28 days atrecommended dosage of 0.126 mg/m²/h or within the range from 0-0.126mg/m²/h to prevent the in vivo formation of tumors from the cell ortissue product delivered to the animal. Dosages of FK866 that arenon-toxic to humans have been described in Holen et al., Invest NewDrugs 26(1):45-51 (2008). The progeny to be delivered to the animal mayinclude any pluripotent stem cell derivative (e.g., cardiomyocyte,neuron, hepatocyte, retinal pigmented epithelial cell).

In another embodiment, the compound is used to eliminate or reduce thenumber of tumorigenic cells from pluripotent stem cell-derivedtumorigenic progeny (defined above) in vitro prior to downstream invitro and in vivo applications. In this method, compound is applied tocell cultures or tissue products for 24-96 hours of treatment time atconcentrations ranging from: 0.1-50 μM for STF-31 and 0.001-10 μM forFK866. After treatment for time required to eliminate the tumorigenicpopulation, the compound is removed by multiple washes of appropriatemedia without compound (e.g., three to five times the volume of culturemedia) and cells are cultured until utilization in downstreamapplication.

1-16. (canceled)
 17. A method of suppressing tumorigenicity of cellsadministered to a subject, the method comprising administering to thesubject in need thereof a cell composition comprising pluripotent stemcell-derived cells contacted to an effective amount of a compound thatselectively reduces or eliminates undifferentiated pluripotent stemcells or partially differentiated pluripotent stem cell derived progeny,wherein the compound is a nicotinamide phosphoribosyltransferase (NAMPT)inhibitor, wherein the pluripotent stem cell-derived cells are contactedto the compound prior to, at the time of, or subsequent toadministration to the subject to suppress tumorigenicity of theadministered cell composition.
 18. (canceled)
 19. The method of claim17, wherein the NAMPT inhibitor is selected from the group consisting ofSTF-31, FK866, GMX-1778, GNE-617, and GNE-618.
 20. The method of claim17, wherein the effective amount is an amount between about 1 nM andabout 100 μM.
 21. The method of claim 17, wherein the effective amountfor contacting at the time of or subsequent to administration to thesubject is between about .1 to 0.126 mg/m²/hour.
 22. The method of claim17, wherein the subject is a human.
 23. The method of claim 22, whereinthe cells are allogeneic or autogeneic to the human subject.
 24. Themethod of claim 17, wherein tumorigenicity is teratoma or tumorformation.
 25. A method of obtaining a cell composition having reducedtumorigenicity for transplantation into a subject, the method comprisingthe steps of: (a) providing a population of pluripotent stemcell-derived cells; and (b) contacting the population to an effectiveamount of a compound that selectively reduces or eliminatesundifferentiated pluripotent stem cells or partially differentiatedpluripotent stem cell derived progeny, wherein the compound is anicotinamide phosphoribosyltransferase (NAMPT) inhibitor, whereby thecontacted population has reduced tumorigenicity relative to apluripotent stem cell-derived cell population not contacted to thecompound, and using the contacted population as a cell population fortransplantation into the subject.
 26. (canceled)
 27. The method of claim25, wherein the NAMPT inhibitor is selected from the group consisting ofSTF-31, FK866, GMX-1778, GNE-617, and GNE-618.
 28. The method of claim25, wherein the effective amount is an amount between about 1 nM andabout 100 μM.
 29. The method of claim 28, wherein the effective amountis about 5 μM STF-31.
 30. The method of claim 28, wherein the effectiveamount is about 25 nM FK866.
 31. The method of claim 25, furthercomprising administering the cell composition having reducedtumorigenicity to the subject.
 32. The method of claim 25, wherein thesubject is a human.
 33. The method of claim 32, wherein the cells areallogeneic or autogeneic to the human subject.
 34. The method of claim25, wherein tumorigenicity is teratoma or tumor formation.